CN103773819B - A kind of lactic acid of producing is coupled simultaneously and prepares the method for GABA - Google Patents
A kind of lactic acid of producing is coupled simultaneously and prepares the method for GABA Download PDFInfo
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- 239000004310 lactic acid Substances 0.000 title claims abstract description 29
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 29
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 26
- 229960003692 gamma aminobutyric acid Drugs 0.000 title claims abstract description 25
- 108091022930 Glutamate decarboxylase Proteins 0.000 claims abstract description 51
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- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims abstract description 10
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- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a kind of lactic acid of producing is coupled simultaneously and prepares the method for GABA, comprise: Rhizopus oryzae is accessed in fermentation medium and fermented, in the time that the pH of zymotic fluid is less than 4.8, in zymotic fluid, add glutamate decarboxylase, phosphopyridoxal pyridoxal phosphate and Pidolidone sodium, continue afterwards to add Pidolidone sodium in zymotic fluid, utilize glutamate decarboxylase catalysis glutamic acid to generate the vigor of GABA, consume the H in environment+, make the pH of zymotic fluid maintain 4.8～6.0, reach and separate that deacidification suppresses lactic acid fermented inhibition, the object of realize lactic acid and GABA time production. A kind of method that the present invention provides new releasing substrate to suppress for lactic fermentation, and coupling produces functional compounds GABA during the fermentation, has not only increased the output of lactic acid, and has improved the economy of process.
Description
Technical field
The invention belongs to Biochemical Engineering field, relate in particular to a kind of lactic acid preparation that is simultaneously coupled of producingThe method of GABA.
Background technology
Lactic acid is a kind of important raw material of industry, is widely used in for a long time food, medicine, changeAdornment product, agricultural and chemical field. In addition, lactic acid can be used for preparing the engineering materials such as PLA. AndPLA can be used to manufacture completely biodegradable plastics, produces environmentally friendly packing material and agricultural thinFilm, the environmental crisis of being brought to eliminate " white pollution ". In addition, because PLA has goodBiocompatibility, is also widely used in medical industry field, comprises suture, medicament slow release materialMaterial, bone fixed repairing material and other used in tissue engineering material etc. Along with market is to PLA demandContinuous increase, also further promoted the continuous increase of market to lactic acid demand. Therefore, exploitationProduction of lactic acid technology is paid close attention to for a long time always widely efficiently.
The production of lactic acid mainly contains chemical synthesis and fermentation method, compared with chemical synthesis, due to lifeThe spy that abundant raw materials, operating temperature are low, energy consumption is low and the optical purity of product is high of thing fermentation methodPoint, and be widely adopted. At present, global lactic acid is all mainly to produce by fermentation method(Abdel-Rahmanetal., 2011). But, for lactic fermentation, because lactic acid causesProduct suppresses to have become the Main Bottleneck problem of effective fermentation. In lactic fermentation process, constantly generateLactic acid zymotic fluid pH is reduced gradually, lower pH value is to thalline normal growth and acid processCan produce strong inhibition, therefore must remove product by the pH in controlled fermentation process and suppress.
At present, conventional pH control method is to add alkaline matter as various carbonate, hydroxideAnd ammoniacal liquor etc., wherein CaCO3It is the most frequently used nertralizer. But, using CaCO3As inWith in lactic fermentation, produce lactic acid time, a large amount of CaCO in fermentation tank3Easily make material taking mouth stop up;In the fermentation middle and later periods, the calcium lactate of high concentration makes zymotic fluid become sticky, generates a large amount of foams, causesEscape liquid; Or occur to solidify phenomenon, make fermentation be difficult to continue. In addition, use CaCO3As nertralizerThere is extraction process long flow path, consume the raw material of industry mainly with and the rate of recovery low, produce a large amount of sulfuric acid simultaneouslyCalcium waste residue and waste water, environmental protection pressure is large, increases processing cost, the problems such as energy consumption height.
Therefore, developing the method that in new releasing lactic fermentation process, product suppresses replaces traditionalCaCO3Neutralisation is of great practical significance.
Summary of the invention
The invention provides a kind of lactic acid of producing and be coupled simultaneously and prepare the method for GABA, for lactic acid is sent outA kind of method that ferment provides new releasing substrate to suppress, and can also be coupled during the fermentationPrepare functional compounds GABA.
Produce lactic acid and be coupled simultaneously and prepare a method of GABA, it is characterized in that, comprising:
Rhizopus oryzae is accessed in fermentation medium and fermented, in the time that the pH of zymotic fluid is less than 4.8,In zymotic fluid, add glutamate decarboxylase, phosphopyridoxal pyridoxal phosphate and Pidolidone sodium, continue afterwards to sending outThe pH that adds Pidolidone sodium to maintain zymotic fluid in ferment liquid is 4.8～6.0.
Glutamate decarboxylase (Glutamatedecarboxylase, GAD; EC4.1.1.15) canUnder acid condition, by consuming H+, the decarboxylation of catalysis Pidolidone generates GABA(γ-aminobutyricacid, GABA). Hypotensive, diuresis, anticonvulsion, pre-that GABA hasAnti-epilepsy, improve sleep, antidepression, promotion hormone secretion and protect the liver the different physiological roles such as sharp kidney.In addition, GABA can be used as that precursor substance comes synthesising biological degradation material polyamide-4 and environment hasThe chemical products such as good plastic solvent N-pyrrolidones. The present invention is by lactic fermentation and glutamic acid decarboxylationThe decarboxylic reaction of enzyme is coupled, and utilizes the decarboxylic reaction of glutamate decarboxylase to consume lactic acid and sends outThe H producing in ferment process+, the product of removing in lactic fermentation process suppresses, and realizes lactic acid simultaneouslyWith GABA time, produce. The key reaction occurring in fermentation is:
In addition, the pH that the present invention maintains zymotic fluid by the decarboxylic reaction of glutamate decarboxylase is4.8～6.0, under this pH condition, be conducive to the carrying out of fermentation reaction, produce a large amount of lactic acid, withTime this pH condition also ensured carrying out smoothly of decarboxylic reaction, press down to remove product in sweatSystem.
When lactic fermentation, it is fermentative microorganism that the present invention adopts Rhizopus oryzae, and the acid resistance of Rhizopus oryzae is stronger,In lactic fermentation process, concrete, can be Rhizopus oryzae As3.819.
Phosphopyridoxal pyridoxal phosphate, as the coenzyme of glutamate decarboxylase, is trembled by add phosphoric acid pyrrole in zymotic fluidAldehyde, can avoid the inactivation at long-time, multiple batches of sweat Glutamic Acid decarboxylase, promotes paddyPropylhomoserin decarboxylation, promotes the generation of GABA, and the final concentration of described phosphopyridoxal pyridoxal phosphate is0.01～1mM, is preferably 0.1mM.
In order to improve the stability of glutamate decarboxylase in system, simplify the later separation of enzyme, described inGlutamate decarboxylase add front through immobilization processing. The method of immobilization processing specifically can adopt routineMeans.
The source formation of described immobilized glutamate decarboxylase can be the glutamic acid decarboxylation after purifyingEnzyme, the crude enzyme liquid that contains glutamate decarboxylase or expression have the cell of glutamate decarboxylase. For avoiding enzymeExtraction and purification step cost, utilization rate and the stability of raising enzyme, preferred, with recombinant expressedThere is the mode of the somatic cells of glutamate decarboxylase to add.
Described somatic cells is preferably restructuring glutamate decarboxylase engineering bacteria, this restructuring glutamate decarboxylaseEngineering bacteria is the Escherichia coli that proceed to glutamic acid decarboxylase gene. (be generally by technique for gene engineeringFirst glutamic acid decarboxylase gene is connected into expression vector, builds and obtain recombinant expression carrier, then will weighGroup expression vector proceeds in Escherichia coli; Wherein, expression vector can be pET-28a, pET-29,PET-30, pET-34, pRSET etc. ) glutamic acid decarboxylase gene is shown in Escherichia coliReach, can effectively improve the catalytic activity of thalline.
Described Escherichia coli be specifically as follows (E.coli) BL21, Escherichia coli (E.coli) BLR,Escherichia coli (E.coli) Origami, Escherichia coli (E.coli) NovaBlue or Escherichia coli (E.coli)Rosetta etc.
Preferably, described glutamic acid decarboxylase gene is derived from Lactobacillus brevis (Lactobacillusbrevis)CGMCC1306. The transcription product of the glutamic acid decarboxylase gene (gadB) of this bacterial classification has betterHeat endurance.
Although can improve the catalysis activity of glutamate decarboxylase by technique for gene engineering, thallineThe barrier action of cell membrane and cell membrane, has limited the transmission inside and outside cell of substrate and product, makesGlutamate decarboxylase vigor in thalline born of the same parents is difficult to give full play to, therefore, and for improving cell membrane and thinThe resistance to mass tranfer of after birth to substrate, can be to expressing the carrying out property of Escherichia coli that has glutamate decarboxylaseChange, the method for described permeability is: the restructuring glutamate decarboxylase work that expression is had to glutamate decarboxylaseJourney bacterium thalline is dissolved in buffer solution, processes 25～35min for 65～75 DEG C and can (be preferably 70 DEG C of processing30min). The glutamate decarboxylase engineering bacteria of permeability can adopt known immobilization intact cellBeing fixed of method.
Immobilized glutamate decarboxylase addition is that every liter of zymotic fluid adds quite total glutamic acid decarboxylationEnzyme activity is the immobilised enzymes of 1500U～5000U. Preferably, add with every liter of zymotic fluid quite totalGlutamate decarboxylase vigor is the immobilised enzymes of 3000U.
For reaching suitable lactic fermentation condition, the temperature of described fermentation is 25～37 DEG C, and rotating speed is160～220r/min; Preferred 200r/min, the temperature of described fermentation is 32 DEG C, rotating speed is 200r/min。
The formula of described fermentation medium is: glucose, 120g/L; (NH4）2SO4，2g/L；KH2PO4，0.14g/L；NaH2PO4，0.16g/L；MgSO4·7H2O，0.25g/L；ZnSO4·7H2O，0.11g/L；CaCO3，5g/L。
Compared with prior art, beneficial effect of the present invention is:
The invention provides in a kind of lactic fermentation process and remove the new method that substrate suppresses, the method canTo have avoided use nertralizer CaCO3Remove bring to zymotechnique of lactic acid when product suppresses unfavorableImpact (as escaping liquid, obstruction material taking mouth etc.).
In addition, the present invention, in the time removing lactic fermenting products inhibition, has realized functional compounds GABAIn time, produces, and reduces the production cost of the two, has also overcome because of CaCO3Add produce a large amount ofCalcium sulfate waste residues and waste water etc., more environmental protection.
The product content that method of the present invention obtains is high, and the content of Lactic Acid from Fermentation Broth can 76～79g/L,The content of GABA can reach 37～40g/L simultaneously.
Detailed description of the invention
Further explain the present invention below in conjunction with detailed description of the invention.
Bacterial classification: Rhizopus oryzae As3.819; Culture presevation slant medium: PDA culture medium.
Engineering bacteria E.coliBL21(DE3)/pET28a-gadB: glutamic acid decarboxylase gene (gadB)Be derived from Lactobacillus brevis (Lactobacillusbrevis) CGMCC1306.
Seed culture medium: glucose, 120g/L; (NH4）2SO4，4g/L；KH2PO4，0.15g/L；ZnSO4·7H2O，0.22g/L；MgSO4·7H2O，0.45g/L。
Fermentation medium: glucose, 120g/L; (NH4）2SO4，2g/L；KH2PO4，0.14g/L；NaH2PO4，0.16g/L；MgSO4·7H2O，0.25g/L；ZnSO4·7H2O，0.11g/L；CaCO3，5g/L。
LB culture medium: dusty yeast, 5g/L; Tryptone, 10g/L; NaCl, 10g/L.
An enzyme activity unit is defined as under 37 DEG C of conditions, and 1min generates 1 μ molGABA(1U=1 μ molGABAin1minat37 DEG C) required enzyme amount.
The ratio vigor of GAD is defined as the enzyme activity unit (Umg that every milligram of dry weight cell possesses-1cells，dryweight）。
Embodiment 1
The preparation of immobilized glutamate decarboxylase engineering bacteria:
(1) engineering bacteria (E.coli of picking glutamate decarboxylase from the LB solid medium of activationBL21(DE3)/pET28a-gadB, (can be referring to " Cloning, sequencingandexpressionofaglutamatedecarboxylasegenefromtheGABA-producingstrainLactobacillusbrevisCGMCC1306[J].ANNALSOFMICROBIOLOGY.2012,62 (2): 689-698 ") access of monoclonal thalline is containing kanamycins (50 μ gmL-1) LBIn fluid nutrient medium, 37 DEG C, 200r/min shaken cultivation is spent the night, and obtains seed liquor.
(2) by seed liquor by volume mark 1% inoculum concentration be inoculated into and contain kanamycins (50μg·mL-1) LB culture medium, be placed in 37 DEG C, 200r/min is cultured to OD600Within 0.6 o'clock, addIPTG, making IPTG final concentration is 0.5 μ M, 30 DEG C, 6h is cultivated in 150r/min induction.
(3) expression there is is the thalline of glutamate decarboxylase process after 30min at 70 DEG C of temperature, fromThe heart is collected thalline, obtains glutamate decarboxylase (GAD) engineering bacteria of permeability, now permeabilityThe catalysis activity of GAD engineering bacteria is 8.35U/mg.
(4) the GAD engineering bacteria of permeability is joined to 15gL-1Sodium alginate soln in mixEvenly, making cell density is 9mgmL-1(dry cell weight), and then mixed liquor is dropwise splashed intoTo 20gL-1Calcium chloride solution in, 4 DEG C are spent the night. With sterilized water washing immobilized thallus 3 times,Be placed in 4 DEG C for subsequent use.
Embodiment 2
(1) inoculation 2%(v/v) Rhizopus oryzae As3.819 spore suspension (5 × 106Individual/mL) inIn seed culture medium, 30 DEG C, shaken cultivation 24 hours on 220r/min shaking table, obtains seed liquor.
(2) with 4% inoculum concentration, seed liquor is seeded in 50mL fermentation medium, at 32 DEG C,Under the condition of 200r/min, cultivate, when zymotic fluid pH is lower than 4.8 time, fixing to adding in zymotic fluid(somatic cells dry weight content is 23.2mg to the glutamate decarboxylase engineering bacteria of changing, and glutamate decarboxylase is totalVigor is 150U), the final concentration phosphopyridoxal pyridoxal phosphate that is 0.1mM, constantly add Pidolidone simultaneouslySodium, maintains between 4.8-5.0 the omnidistance pH value of zymotic fluid, continues to cultivate 60 hours. Simultaneously withThe zymotic fluid of the glutamate decarboxylase of being fixed not is blank.
The content of coupling process Lactic Acid from Fermentation Broth is 79g/L after measured, and the content of GABA is40g/L. And lactic acid production 39.49g/L in blank articles for use, and GABA can't detect.
Embodiment 3
(1) inoculation 2%(v/v) Rhizopus oryzae spore suspension (5 × 106Individual/mL) in seed cultureIn base, 30 DEG C, shaken cultivation 24 hours on 220r/min shaking table, obtains seed liquor.
(2) with 4% inoculum concentration, seed liquor is seeded in 50mL fermentation medium, at 32 DEG C,Under the condition of 200r/min, cultivate, when zymotic fluid pH is lower than 4.8 time, fixing to adding in zymotic fluid(somatic cells dry weight content is 23.2mg to the glutamate decarboxylase thalline of changing, and glutamate decarboxylase is always livedPower is 150U), the phosphopyridoxal pyridoxal phosphate that final concentration is 0.1mM, constantly adds Pidolidone sodium simultaneously,The omnidistance pH value of zymotic fluid is maintained between 5.2-5.6, continue to cultivate 60 hours. Simultaneously not addThe zymotic fluid of immobilized glutamate decarboxylase is blank.
The content of Lactic Acid from Fermentation Broth is 76g/L after measured, and the content of GABA is 37g/L. And it is emptyLactic acid production 36g/L in white contrast articles for use, and GABA can't detect.
Embodiment 4
(1) inoculation 2%(v/v) Rhizopus oryzae spore suspension (5 × 107Individual/mL) in seed cultureIn base, 30 DEG C, shaken cultivation 24 hours on 220r/min shaking table, obtains seed liquor.
(2) with 5% inoculum concentration, seed liquor is seeded in 50mL fermentation medium, at 32 DEG C,Under the condition of 220r/min, cultivate, when zymotic fluid pH is lower than 5.0 time, fixing to adding in zymotic fluid(somatic cells dry weight content is 23.2mg to the glutamate decarboxylase engineering bacteria of changing, glutamate decarboxylaseTotal activity is 150U), the final concentration phosphopyridoxal pyridoxal phosphate that is 0.1mM, constantly add L-paddy ammonia simultaneouslyAcid sodium, maintains between 5.0-5.2 the omnidistance pH value of zymotic fluid, continues to cultivate 60 hours. SimultaneouslyTaking the zymotic fluid of the glutamate decarboxylase of being fixed not as blank.
The content of coupling process Lactic Acid from Fermentation Broth is 77g/L after measured, and the content of GABA is41g/L. And lactic acid production 38g/L in blank articles for use, and GABA can't detect.
Claims (2)
1. produce lactic acid and be coupled simultaneously and prepare a method of GABA, it is characterized in that, comprising:
Rhizopus oryzae is accessed in fermentation medium and fermented, in the time that the pH of zymotic fluid is less than 4.8,In zymotic fluid, add glutamate decarboxylase, phosphopyridoxal pyridoxal phosphate and Pidolidone sodium, continue afterwards to sending outThe pH that adds Pidolidone sodium to maintain zymotic fluid in ferment liquid is 4.8～6.0;
The final concentration of described phosphopyridoxal pyridoxal phosphate is 0.01～1mM; The temperature of described fermentation is 25～37 DEG C;
Described glutamate decarboxylase adds front through immobilization processing; Adding of immobilized glutamate decarboxylaseDosage is: every liter of zymotic fluid adds suitable glutamate decarboxylase total activity, and to be 1500U～5000U consolidateSurely change enzyme;
The source formation of described glutamate decarboxylase is glutamate decarboxylase after purifying, de-containing glutamic acidThe crude enzyme liquid of carboxylic acid or expression have the somatic cells of glutamate decarboxylase; Described glutamate decarboxylaseGENE SOURCES is from Lactobacillus brevis (Lactobacillusbrevis) CGMCCNO.1306.
2. the method for claim 1, is characterized in that, described somatic cells is glutamic acidDecarboxylase engineering bacteria, this glutamate decarboxylase engineering bacteria is the large intestine bar that proceeds to glutamic acid decarboxylase geneBacterium.
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